The gut microbiome plays a critical role in the development of the immune system and alterations in the microbiome diversity almost certainly play an important role in the surge of allergic and noncommunicable inflammatory diseases in developed nations. The infant gut microbiome develops over the first 2 years of life, and the WHEALS birth cohort showed that breastfeeding is the most significant factor impacting infant gut microbiome composition. However, breast milk has several bioactives that can modulate microbial composition and are not captured in a coarse definition of breastfeeding. Human milk oligosaccharides (HMOs) are complex glycans, non-digestible by humans, and one of the main substrates for the infant gut microbiota until weaning. Depending on the Lewis blood group, secretor status and diet, mothers' milk has significant variations in qualitative and quantitative HMO composition. Furthermore, breast milk has also been shown to act as a source of bacterial species that colonize the infant gut. Finally, IgA and IgA-inducing cytokines reflect maternal exposures and also control gut microbiome composition. These findings support a strong role for breast milk in determining early life microbial composition. However, human milk composition varies significantly between mothers, and the relative contribution of these factors on infant gut microbiome is, however, unknown. The overall objective of this application is to define how human milk bioactives support the development of infant gut microbiome and metabolome. Our central hypothesis is that life-style factors related to modern lifestyle increase the risk for dysbiotic microbiome and promote allergies, in part mediated via lack of positive modulation by breast milk. Our preliminary data from the Old Order Mennonites (OOM) with rich exposure to farms and unpasteurized milk suggest that they have a low prevalence of asthma and allergic diseases and breast milk with a more diverse microbiome and high levels of IgA. We will determine breast milk bioactive components in a diverse population of mothers with varying exposure (Aim 1) and characterize the composition of infant gut microbiome and metabolome and assess the contribution of breast milk composition to infant gut microbiome and metabolome (Aim 2). The proposed project is founded on our CTSI-funded OOM pilot study in which breast milk and stool samples were collected at 1-2 months from breastfeeding OOM (n=58) and Rochester (n=34) mother-infant pairs. We expect to identify the key human milk components that impact the development of infant gut microbiome and metabolome. The ultimate goal is to establish maternal interventions to modify breast milk composition to favor protection against allergic and non-communicable inflammatory diseases.